Project 1: Biochemistry of p66Shc and its role in adipose tissue The chemical and biochemical function of p66Shc underlies all of the consequences of p66Shc depletion, which include primarily the perturbed pathways and their participation in lifespan extension. The Pelicci group has recently establishedythat p66Shc has a reduction-oxidation (redox) function, and that the redox function appears to underlie all'of the biochemical and biological properties of p66Shc that have been demonstrated to date, including reactive oxygen species (ROS) generation and apoptosis. It is clear that there is p66Shc in both the mitochondrial and cytoplasmic compartments, which may have similar or different physiological roles. In mitochondria, it is thought that p66Shc produces apoptogenic H2O2 that mediates apoptosis. In the cytoplasm and plasma membrane, the redox function of p66Shc may be important to the NADPH oxidase-mediated respiratory burst, adipogenesis, and life-shortening metabolic syndrome. Thus, the overall goals of this project are focused on identifying the proteins that interact with p66Shc in the mitochondria, how they arrive there, and the underlying biochemical functions of p66Shc in both the mitochondrial and cytoplasmic compartments, specifically with respect to metabolic syndrome. The specific aims of this project are the following: (1) Identify the proteins that interact with p66Shc in the mitochondria and perform a functional analysis of p66Shc protein import into mitochondria; by identifying proteins that interact with p66Shc, we will be more likely to determine the physiological roles of p66Shc. (2) Characterize the reaction between p66Shc and cytochrome c; it is likely that the interaction between p66Shc and cytochrome c controls the pro-apoptotic function of p66Shc. (3) Understand the function of p66Shc in fat tissue; strong preliminary data demonstrate a role for p66Shc in adipogenesis specifically at the step of differentiation, and we will determine the mechanism by which this is inhibited. (4) Validate p66Shc as a contributor to metabolic syndrome; obesity and age are main contributors to the development of 'metabolic syndrome,' and it has recently been demonstrated that NADPH oxidase plays a critical role in the generation of H2O2 in this syndrome. Thus, we plan to investigate the interaction of p66Shc with the other components of the metabolic syndrome pathway. Relevance to public health: It is clear that a mechanistic understanding of the physiological function of p66Shc depletion, which reduces fat, extends lifespan, and may reduce metabolic syndrome, is likely to be relevant to public health.